Human immunodeficiency virus (HIV) entry is known to require a complex interaction of the viral envelope glycoprotein (Env) with CD4 and cellular chemokine receptors. HIV Env protein is produced as a precursor (gp160) that is subsequently cleaved into two parts, gp120 which binds CD4 and chemokine receptors, and gp41 which is anchored in the viral membrane and mediates membrane fusion. Differential use of chemokine receptors by HIV and simian immunodeficiency virus (SIV) has largely explained differences in tropism among different isolates (Berger, 1997, AIDS 11:S3-S16; Hoffman and Doms, 1998, AIDS 12:S17-S26). While a number of chemokine receptors can be utilized by HIV or SIV (Deng et al., 1997, Nature 388:296-300; Choe et al., 1996, Cell 85, 1135-1148; Rucker et al., 1997, J. Virol. 71:8999-9007; Edinger et al., 1997, Proc. Natl. Acad. Sci. USA 94:14742-14747; Liao et al., 1997, J. Exp. Med. 185:2015-2023; Farzan et al., 1997, J. Exp. Med. 186:405-411), CCR5 and CXCR4 appear to be the principal coreceptors for HIV-1 (Zhang et al., 1998, J Virol. 72:9337-9344; Zhang et al., 1998, J. Virol. 72:9337-9344). Isolates of HIV that first establish infection target blood lymphocytes and macrophages using CCR5 (Alkhatib et al., 1996, Science 272:1955-1958; Deng et al., 1996, Nature 381:661-666; Dragic et al., 1996, Nature 381:667-673; Doranz et al., 1996, Cell 85:1149-1158), while viruses that are generally associated with progression to AIDS and can infect T cell lines in vitro use CXCR4 (Choe et al., 1996, Cell 85:1135-1148; Feng et al., 1996, Science 272:872-876; Connor et al., 1997, J. Exp. Med. 185:621-628).
Binding of Env to CD4 initiates poorly understood conformational changes enabling gp120 to bind to a chemokine receptor and leading to fusion of the viral and cellular membranes (Jones et al., 1998, J. Biol Chem. 273:404-409; Moore et al., 1994, J. Virol. 68:469-484; Wyatt, 1992, J. Virol. 66:6997-7004; Wu et al., 1996, Nature 384:179-183). Thus, the Env glycoproteins gp120 and gp41 are important potential targets for neutralizing antibodies to HIV and SIV. As stated previously, Env is a protein structural component comprising the retroviral capsid, and is produced from a precursor molecule (gp160) that is cleaved in the Golgi, transported to the cell surface, and incorporated into virions as trimers of noncovalently associated gp120/gp41 subunits (Allan et al., 1985, Science 228:1091-1094; Chan et al., 1997, Cell 89:263-273; Earl et al., 1990, Proc. Nat. Acad. Sci. 87:648-652; Rizzuto et al., 2000, AIDS Res. Hum. Retroviruses 16:741-9; Pinter et al., 1989, J. Virol. 63:2674-2679; Robey et al., 1985, Science 228:593-595). Gp120 is extensively glycosylated and contains 5 conserved and 5 hypervariable regions. Four of the hypervariable regions, designated V1, V2, V3 and V4 are loops formed by intramolecular disulfide bonds and exposed on the protein surface (Modrow et al., 1987, J. Virol. 61:570-578; Starcich et al., 1986, Cell 45:637-648). In HIV-1, V1 extends from the V2 loop while in HIV-2 and SIV a more complex loop structure exists containing two additional disulfide bonds (Hoxie et al., 1991, AIDS Res Hum Retroviruses 7:495-9). The conserved regions on gp120 fold into a core structure containing a recessed cavity that forms a CD4 binding site (CD4bs) and a “bridging sheet” that connects an inner and outer domain and largely forms a coreceptor binding site for CCR5 and CXCR4 (Basmaciogullari et al., 2002, J. Virol. 76:10791-800; Kwong et al., 2000, Structure Fold Des. 8:1329-39; Kwong et al., 1998, Nature 393:648-659; Rizzuto et al., 1998, Science 280:1949-1953; Wyatt et al., 1998, Nature 393:705-711). Conserved regions on the gp120 core also likely abut gp41 in the Env trimer and are exposed only if it dissociates from gp41 (Kwong et al., 2000, J. Virol. 74:1961-1972). Gp41 contains two heptad repeat regions, HR1 and HR2, and a hydrophobic amino-terminal fusion peptide required to initiate lipid mixing between viral and cell membranes (Martin et al., 1996, J. Virol. 70:298-304; Pereira et al., 1997, Biophys. J. 73:1977-86; Weng et al., 2000, J. Virol. 74:5368-5372; Wild et al.,1994, Proc. Natl. Acad. Sci. USA 91:9770-4).
Cell entry by HIV and SIV is initiated by an interaction of gp120 with CD4, leading to extensive conformational changes that, measured on monomeric gp120, are associated with a loss of entropic freedom (Myszka et al., 2000, Proc. Natl. Acad. Sci U.S.A. 97:9026-903 1), movement of hypervariable loops V1/V2 and V3 (Moore et al., 1993, J. Virol. 67:6136-6151; Sattentau et al., 1993, J. Virol. 67:7383-7393; Wyatt et al., 1995, J. Virol. 69:5723-5733; Wyatt et al., 1993, J. Virol. 67:4557-4565), and the exposure and/or formation of the bridging sheet (Myszka et al., 2000, Proc. Natl. Acad. Sci U.S.A. 97:9026-9031). V3 and particularly the β19 strand within the bridging sheet likely bind to the chemokine receptor and, at least for CCR5, create a high affinity interaction (Cormier et al., 2002, J. Virol. 76:8953-7; Dragic et al., 2001, J. Gen. Virol. 82:1807-14; Farzan et al., 1999, Cell 96:667-76; Trkola et al., 1996, Nature 384:184-7). V3 mediates specificity (i.e., determines whether CXCR4 or CCR5 are utilized) and likely interacts with extracellular loops of chemokine receptors, while the bridging sheet likely interacts with both receptors, and at least for CCR5 probably binds to the N-terminus (Basmaciogullari et al., 2002, J. Virol. 76:10791-800; Dragic et al., 2001, J. Gen. Virol. 82:1807-14; Farzan et al., 2002, J. Biol. Chem. 277:40397-402; Farzan et al., 2000, J. Biol. Chem. 275:33516-21; Rizzuto et al., 2000, AIDS Res. Hum. Retroviruses 16:741-9; Rizzuto et al., 1998, Science 280:1949-1953). Subsequent to or concurrent with chemokine receptor binding, the gp41 fusion peptide inserts into the membrane of the cell, and gp41 undergoes a conformational rearrangement in which HR1 and HR2, in the context of a trimer, associate in an antiparallel manner to form a highly stable six helix bundle, thereby bringing the viral and cell membranes into close proximity and inducing membrane fusion (Matthews et al., 1994, Immunol. Rev. 140:93-104; Melikyan et al., 2000, J. Cell Biol. 151:413-23). Thus, beginning with CD4 engagement, gp120 and gp41 undergo a highly coordinated sequence of events that involve extensive conformational changes and inter- and intra-molecular interactions as chemokine receptors are engaged and viral and cell membranes are brought together.
Given the complexity of viral entry and the numerous steps that could be blocked by antibody binding, it is remarkable that the humoral response in infected hosts fails to arrest this process. Initial antibody responses are directed against epitopes that are revealed only on dissociated gp120 monomers and exhibit limited or no reactivity with Env trimers (Parren et al., 1999, AIDS 13:S137-S162; Wyatt et al., 1998, Nature 393:705-711). Although neutralizing antibodies are produced within one month after infection, these are type-specific and directed primarily against variable loops V1/V2 and V3, which can tolerate extensive genetic changes, and viral escape mutants are rapidly generated (Richman et al., 2003, Proc. Nat. Acad. Sci. USA 100:4144-9). Broadly neutralizing antibodies are either not produced or are produced only late after infection and in low titer (Richman et al., 2003, Proc. Nat. Acad. Sci. USA 100:4144-9; Wyatt et al., 1998, Science 280:1884-1888). The basis for HIV's neutralization resistance likely arises from a number of structural attributes of Env, and in particular a lack of exposure, accessibility or immunogenicity of functionally important epitopes on the assembled Env trimer (Fouts et al., 1997, J. Virol. 71:2779-85; Kwong et al., 2000, Structure Fold Des 8:1329-39; Parren et al., 1999, AIDS 13:S137-S162; Sullivan et al., 1998, J. Virol. 72:6332-8). First, as noted above, substantial portions of surface exposed regions on gp120 contain N-linked carbohydrates, which are poorly immunogenic and capable of masking underlying domains, a property initially termed “carbohydrate cloaking” (Kwong et al., 2000, Structure Fold Des 8:1329-39) and more recently, the “glycan shield” (Wei et al., 2003, Nature 422:307-12). Second, gp120 undergoes extensive thermodynamic changes following CD4 binding with a large increase in enthalpy (ΔH) and a decrease in entropy (ΔS), reflecting increased molecular ordering and an extensive loss of conformational flexibility (Myszka et al., 2000, Proc. Natl. Acad. Sci U.S.A. 97:9026-903 1). It has been proposed that the intrinsic flexibility of gp120 prior to CD4 triggering could in itself mask epitopes for broadly neutralizing antibodies (Kwong, et al., 2002, Nature 420:678-82; Myszka et al., 2000, Proc. Natl. Acad. Sci U.S.A. 97:9026-903 1). Third, although crystallographic resolution of variable loops has not been achieved, two critical functional domains, the CD4bs and bridging sheet, are flanked by the V1/V2 and V3 loops, which are well positioned to restrict access to these conserved functional domains prior to CD4 triggering. Fourth, there are likely to be additional steric constraints on antibody binding to core domains in the context of an oligomeric Env trimer during its interaction with CD4 and chemokine receptors on target cell surface. Indeed, for some human monoclonal antibodies to CD4-induced epitopes that partially overlap the bridging sheet, their neutralizing activity is markedly enhanced as Fab and single chain (scFv) fragments compared to their intact immunoglobulins (Labrijn et al., 2003, J. Virol. 77: In Press; Moulard et al., 2002, Proc. Natl. Acad. Sci. USA 99:6913-8).
Despite these obstacles, anti-HIV-1 Env human monoclonal antibodies have been characterized that exhibit, to varying degrees, broadly neutralizing activity. These include b12, reactive with the CD4bs (Kessler, et al., 1997, AIDS Res. Hum. Retroviruses 13:575-582); 17b, 48d, X5, and others reactive with CD4-induced epitopes on the gp120 core (Moulard et al., 2002, Proc. Natl. Acad. Sci. USA 99:6913-8114; Xiang et al., 2002, AIDS Res Hum Retroviruses 18:1207-17); 2G12, reactive with an exposed conformational epitope on gp120 determined by high mannose carbohydrates (Calarese et al., 2003, Science 300:2065-71; Trkola et al., 1996, J. Virol. 70:1100-1108); and 2F5 and other monoclonal antibodies reactive with linear epitopes on the membrane proximal region of gp41 (Muster et al., 1993, J. Virol. 67:6642-6647; Parker et al., 2001, J. Virol. 75:10906-11; Zwick et al., 2001, J. Virol. 75:10892-905). As noted above, passive administration of combinations of these antibodies has protected animals from mucosal and parenteral challenges with pathogenic SHIVs (Baba et al., 2000, Nature Med. 6:200-206; Mascola et al., 1999, J. Virol. 73:4009-4018; Mascola et al., 2000, Nat. Med. 6:207-210; Ruprecht et al., 2003, Vaccine 21:3370-3). Recent studies have provided insights into remarkable structural attributes of some of these antibodies that contribute to their neutralizing activity including 1) extended CDR3 loops that can access recessed domains (Choe et al., 2003, Cell 114:161-70; Saphire et al., 2001, Acta. Crystal. D. Biol. Crystal. 57:168-71); 2) novel conformational rearrangements in heavy and light chain domains that increase the number of contact sites (Calarese et al., 2003, Science 300:2065-71); 3) variable domains that mimic CD4 (Saphire et al., 2001, Acta. Crystal. D. Biol. Crystal. 57:168-71); and 4) tyrosine sulfation at their antigen binding sites that likely mimics the sulfated N-terminus of CCR5 (Choe et al., 2003, Cell 114:161-70). Although the challenge of generating such antibodies with vaccine preparations may seem daunting, the monoclonal antibodies noted above were all derived from infected humans, and thus provide a strong indication that native immune responses to HIV exist that will produce broadly neutralizing antibodies when immunogens are designed that elicit them.
Given the failure of monomeric gp120 to elicit antibodies that neutralize or even react with native Env trimers of diverse isolates (Parren et al., 1999, AIDS 13:S137-S162), it is likely that Env-based immunogens will need to present relevant epitopes in the context of trimeric Env. Although attempts are underway to stabilize soluble Env trimers (Binley et al., 2000, J. Virol. 74:627-643; Yang et al., 2000, J. Virol. 74:5716-5725; Yang et al., 2002, J. Virol. 76:4634-42) or to present trimers on inactivated viral particles (Lifson et al., 2002, J. Med. Primatol. 31:205-16; Willey et al., 2003, J. Virol. 77:1163-74) or proteoliposomes (Grundner et al., 2002, J. Virol. 76:3511-21), little is known about modifications of Env that can enhance neutralizing antibody responses. Approaches have included gp120s that are deleted of variable loops (Barnett et al., 2001, J. Virol. 75:5526-40; Kim et al., 2003, Virology 305:124-37; Lu et al., 1998, AIDS Res. Hum. Retroviruses 14:151-5; Sanders et al., 2000, J. Virol. 74:5091-5100; Srivastava et al., 2003, J. Virol. 77:2310-20; Stamatatos et al., 1998, AIDS Res. Hum. Retrbviruses 14:1129-1139), deglycosylated (Bolmstedt et al., 2001, Vaccine 20:397-405; Reitter et al., 1998, Nature Med. 4:679-684), bound to CD4 (Dey et al., 2003, J. Virol. 77:2859-65; Fouts et al., 2002, Proc. Natl. Acad. Sci. USA 99:11842-7), or structurally modified to mimic a CD4-bound state (Xiang et al., 2002, J. Virol. 76:9888-99). Given the conserved nature of gp120 core domains between a neutralization-sensitive, lab-adapted isolate and a neutralization-resistant, primary isolate (Kwong et al., 2000, Structure Fold Des 8:1329-39), it is likely that differences in the overlying hypervariable loops play a central role in determining neutralization resistance, providing some rationale for deleting these structures from potential immunogens. Moreover, broadly neutralizing antibodies tend to recognize discontinuous epitopes on the gp120 core while type specific antibodies react with variable loops (Ho et al.,1991, J. Virol. 65:489-493; Posner et al., 1991, J. Immunol. 146:4325-4332; Thali et al., 1992, J. Virol. 66(9):5635; Trkola et al., 1996, Nature 384:184-7; Wu et al., 1996, Nature 384:179-183).
A drawback to genetic or biochemical modifications of gp120 is the potential to disrupt Env structure, ablating relevant epitopes that are exposed during entry. In this regard, Envs have been derived from HIV-1 (Wyatt et al., 1993, J. Virol. 67:4557-4565), SIV (Johnson et al., 2003, J. Virol. 77:375-81; Puffer et al., 2002, J. Virol. 76:2595-605) and SHIVs (Stamatatos et al., 1998, J. Virol. 72:7840-7845) with V1 and/or V2 deletions that remain replication competent, thereby preserving key functional domains. In one study soluble Env from a replication competent SHIV with a V2 deletion elicited a more broadly reactive and qualitatively different humoral immune response with increased reactivity to V3 and C5 domains (Barnett et al., 2001, J. Virol. 75:5526-40). However, this “minimalist” approach to Env modification has been limited by the extent to which Envs retain function and by inference biologically relevant domains after variable loops are deleted (Kim et al., 2003, Virology 305:124-37; Wyatt et al., 1993, J. Virol. 67:4557-4565). Studies with soluble Envs containing more extensive variable loop deletions have been disappointing, likely due to perturbations in Env structure (Kim et al., 2003, Virology 305:124-37; Sanders et al., 2000, J. Virol. 74:5091-5100). Indeed, even partial deletions of the V3 loop (Wyatt et al., 1998, Nature 393:705-711) have resulted in fusion-defective Envs (Wyatt et al., 1995, J. Virol. 69:5723-5733; Wyatt et al., 1993, J. Virol. 67:4557-4565), consistent with its importance in coreceptor binding (Dragic et al., 2001, J. Gen. Virol. 82:1807-14).
HIV is particularly adept in evading humoral immune responses, a feature that likely contributes to the ability of this virus to establish a persistent infection. Although neutralizing antibodies are produced to viral envelope glycoproteins (Env), such antibodies are characteristically directed to hypervariable loops on gp120 (V1/V2 and V3), which can tolerate extensive genetic variation. These antibodies are in general “type specific” and easily circumvented by ongoing viral mutations.
The variable loops also serve to protect domains on the core of gp120, which include highly conserved binding sites for CD4 and chemokine receptors (CCR5 and CXCR4) that are required for entry into target cells. In order for broadly neutralizing antibodies to be produced against HIV, it is likely that these and/or other conserved domains will need to be targeted. A priority for HIV vaccine research efforts is to develop envelope-based immunogens that can elicit these antibodies.
For one simian immunodeficiency virus (SIV) and for HIV-1 Env proteins, it has been shown that V1/V2 can be deleted while preserving replication competence. These V1/V2-deleted viruses have exhibited novel biological properties including CD4-independence, increased neutralization sensitivity, and/or attenuated pathogenicity. In the SIV model, these proteins are under evaluation as vaccine candidates. However, to date, viruses with V3 deletions have not been generated, and it has been generally viewed that the V3 loop is indispensable for viral entry.
It has been an ongoing objective to identify determinants of HIV infectivity as well as determinants that enable it to evade the host immune response in order to gain an understanding of the means by which the virus establishes and maintains infection in the host. Despite the critical nature of the gp120 V1/V2 loops, it has been shown that deletion of the V1/V2 loops from HIV-1 (and SIV) does not abolish viral infectivity. Accordingly, there is a long-felt need to understand the minimal elements of the envelope glycoprotein that are essential for infection, as well as those that are required for immune evasion. Such an understanding is crucial to the development of immunogens capable of eliciting broadly neutralizing antibodies to HIV.
There is an urgent need to develop a vaccine that can prevent HIV infection. Evidence from infected humans and nonhuman primate models suggests both cellular and humoral immune responses can exert at least some control of virus infection in vivo (Amara et al., 2001, Science 292:69-74, Barouch et al., 2000, Science 290:486-92; Borrow et al., 1994, Journal of Virology 68:6103-6110; Egan et al., 2000, J. Virol. 74:7485-95; Gauduin et al., 1997, Nature Med. 3:1389-1393; Jin et al., 1999, J. Exp. Med. 189:991-998; Johnson et al., 2003, J. Virol. 77:375-81; Koup et al., 1994, J. Virol. 68:4650-4655; Kuroda et al., 1999, J. Immunol. 162:5127-5133; Mascola et al., 1999, J. Virol. 73:4009-4018; Mascola et al., 2000, Nat. Med. 6:207-210; Matano et al., 1998, J. Virol. 72:164-9; Parren et al., 2001, J. Virol. 75:8340-7; Schmitz et al., 1999, Science 283:857-860; Schmitz et al., 2003, J. Virol. 77:2165-73; Seth et al., 2000, J. Virol. 74:2502-9), and there is a growing consensus that both will be required to develop a vaccine that either blocks transmission or prevents disease onset (McMichael et al., 2003, Nature Med. 9:874-80). In addition, for protective immunity to be achieved, there is increasing evidence that broadly neutralizing antibodies will be required. Vaccines that elicit a primarily cellular immune response can delay or possibly prevent the onset of disease but in general fail to prevent infection (Barouch et al., 2000, Science 290:486-92; Robinson et al., 1999, Nature Med. 5:526-534; Shiver et al., 2002, Nature 415:331-5). However, in some animal models the level of neutralizing antibodies has correlated with protection from a viral challenge (Berman et al., 1992, J. Virol. 66:4464-9; Emini et al., 1992, Nature 355:728-730; Nishimura et al., 2002, J. Virol. 76:2123-30; Parren et al., 2001, J. Virol. 75:8340-7), and protection from parenteral and mucosal challenges has been achieved by passive administration of neutralizing monoclonal and polyclonal antibodies (Baba et al., 2000, Nature Med. 6:200-206; Mascola et al., 1999, J. Virol. 73:4009-4018; Mascola et al., 2000, Nat. Med. 6:207-210; Poignard et al., 1999, Immunity. 10:431-438; Ruprecht et al., 2003, Vaccine 21:3370-3; Shibata, et al., 1999, Nat. Med. 5:204-210). Unfortunately, while it has become clear that broadly neutralizing antibodies are highly desirable, to date no immunogen has been able to elicit them with any degree of efficiency (McMichael et al., 2003, Nat. Med. 9:874-80). It is therefore crucial for research to address why an infected host fails to produce these antibodies and how vaccines can be designed that will overcome this obstacle.
To date, the ability of HIV-1 to escape the immune system has hindered development of efficacious vaccines to this important human pathogen. Thus, there is a long-felt and unfilled need for the development of effective vaccines and therapeutic modalities for HIV-1 infection in humans. The present invention meets those needs.